Mercury derivative of halogenated fluoresceins



substances, the arbitrary designation a Patented May 24, 1932 l r Tins PATENT 1 oFFrcE',

EDWIN 0. WHITE, 01? BALTIMORE, MARYLAND, ASSIGNOR To Harrison, wEsTooTT AND DUNNING nc, A CORPORATION or MARYLAND MERCURY DERIVATIVE or "HALoGENATED TLUonEscEms No Drawing. Application filed November 18 1925. Serial No. 69,930.

-Mercury derivatives of certain haloge ceins; having most probably thegener'al'for A nated fluoresoeins have been described-but mula: these derivatives are limited as to the nature Z of the halogens, their position inthe fiuores- 5 cein moleoule and their number. This invention relates to mercury derivatives of certain halogenated fiuoresceins which have heretofore not been subjected to the'mercura tion process." The physiological properties of these mercury derivatives vary with the nature, position and number-of the halogen atoms. a

Fluorescein contains three benzene rings, tWo of them ofphenoliccharacter; the other a ring contains'no phenolic group, and will be referred to as the phthalic acid ring. In order to indicate conveniently the positions of the halogensin the hereinafter mentioned oo i wherein X represents hydrogenor halogen,

' drogen or halogen,"two Zs being halogens. 3 III. ,8 dihalogeno -"fluor'esceins, having inost probably the general formula;

will be applied before the names of halogens which lie in the'phenolic rings, and the arbitrary designation ,8 will be applied before the names of halogens which lie in the phthalic acid ring. Thus, -a-di-iodo-,8-tetra-, bromfluorescein signifies a' fluorescein in which two iodine atoms are substituted in the phenolic ringsand fours bromine atoms are substituted in the phthalic acid ring.

The following classes of parentsubstances are included within the scope of the invention: a

I. a-dihalogeno-fiuoresceins, having-most probably the general formula} whereinZ represents hydrogen orhalogen,

- fiuoresceini and 'B-di-iodo-fluorescein.

IV. ,8-tetrahalogeno fluor'esceins, having most probably the general formula:

i Halogen i l I 46 Halogen Hanger 7 H a1oge1i I wherein X represents hydrogen or halogen, H 3 two Xs being halogens,-specifically oc-diClllQ- ro-fluorescein and a-di-iodo-fluoresoein.

specificallyB-tetra brom fluoresceineandi ,8-tet= 50: II. a-dihalogenoe B- dihalogeno -fluores s l F two Zs being halogens, specifically ,B-dibrom-j two Xs' being halogens, and Z represents hycan too

its

5 an alkali solution and l l X7 l X HO OH I I Ha1ogen Halogen wherein X represents hydrogen or halogen, two Xs being halogens. .All combinations of six halogen atoms to the molecule, conforming to this distribution in the several arts of the fluorescein molecule, are specifically included in this group.

These substances may be mercurated by any one of three processes. By the first process the halogenated fluorescein is dissolved in reprecipitated in a finely divided condition by theaddition of an excess of acetic acid. To this suspension is added a suitable amount, but not an eX- cess, of a solution of a mercury salt, preferably mercuric acetate, and the mixture, well stirred, is heated until a sample of the mixture does not blacken with ammonium sulphide solution.

the salt solution evaporated to dryness, preferably under reduced pressure. By the second process the halogenated fiuorescein is dissolved in two. molecular equivalents of standard caustic alkali solution, a suitable amount of yellow mercuric oxide is added to the solution, and with con- 'stant stirring the suspension is heated until all the oxide dissolves. Any insoluble mat: ter which may settle out is removed by filtration and the solution is then evaporated to dryness, preferably under reduced pres-' sure. 7

By the third process the halogenated fluorescein is dissolved in two molecular iequivalents of a standard caustic alkali solution, a suitable amount of a solution of a mercury salt, preferably mercuric acetate, is added with stirring. A precipitate forms, and the mixture is heated-until a sample does not I that is The precipitate is filtered, Washed and dried and converted to an alkali metal salt by solution in caustic alkali, and

oxide used. The meaning of this is as follows: The amount of mercury that can readily enter the rings of a cein depends upon the number of halogen atoms lying in the a-POSltiOIlS. In those fiuoresceins containing no a-halogens, one, two, three or four atoms of mercury can enter the fluorescein molecule, but in those containing two u-halogens only one or two atoms of mercury enter. In any individual case, then, the amount of mercury used will depend upon the degree of. saturation with mercury desired, within the limit set by the number of (it-halogens present.

It is believed that mercury enters the phenolic rings, and presumably 'in positions ortho to the hydroxyl groups, as the mercury derivatives formed dissolve in alkali solutions and do not blacken readily in thec'old, with ammonium sulphide. The susceptibility to blackening with this reagent varies with time of contact and temperature, and Varies among the different mercury derivatives.

halogenated fluores- From the foregoing,therefore, it follows 1 I that the mercury-containing reactionproducts obtained by the mercuration,'by any one I of the three processes just disclosed, of parent substances of the five classes hereinbefore set out, may be represented by the following general formulae, it being understood that said formulae are based upon purely theoretical considerations r I. Mercury derivatives of a-dihalogeno-fiuoresceins having most probably the general formula HO 0H 2 wherein 00 represents hydrogen, halogen or mercury, two aas being halogens and one and not more than two acs being mercury.

II. Merucury derivatives of a-dlhfllOgIlO- B-dihalogeno-fluoresceins having most probably the general formula! wherein 00 represents hydrogen, halogen or mercury, two azsbeing halogens and at least one and not more than two ms being mercury, and .2 represents hydrogen or halogen, two zs being halogens.

7 III. Mercury derivatives of ,B-dihalogenofluoresceins having most probably the general formula wherein 2 represents hydrogen or halogen, two zs being halogens, and y represents hydrogen or mercury, at least one y being mercury, and at least two ys being hydrogen.

IV. Mercury derivatives of ,B-tetrahalogeno-fiuoresceins having most probably the general formula:

Halogen I I O O Halogen \0 Halogen Halogen HO OH 7 wherein 1 represents hydrogen or mercury, at least one y being mercury and at least two ys being hydrogen, and wherein halogen represents bromine or iodine. t i

V. Mercury derivatives of a-(lll12LlOgI10- ,B-tetrahalogeno-fluoresceins having probably the general formula:

Halogen co Halogen 0 Halogen- 7 Halogen I Z I I- 2' HO OH I O I I wherein m represents hydrogen, halogen or mercury, two ws being halogen and at least one and not more than two ws being mercury.

In the foregoing formulae, and in the appended claims, the expression mercury is to be understood as designating a mercury atom most or a 'mercury-containing' grouplwhich most probably is-HgOH. v

Several examples will be given to illustrate the invention. In any example theparticula-r type of reactionused may besubstituted by either of the two alternative processes mentioned above and illustrated in'other exa'm- V ples. Also,-in any example the particular halogenatedfiuorescein may be replaced by the molecular equivalent of any otherone included in the before mentioned severalclasses of parent substances. 1 Example I rescein (O;l gram molecule) are dissolved in 200 c. c. of normal sodi m, hydroxide solution, diluted to about 1' litre, and asolutio'n of 32 grams (0.1 gram molecule) of mercuric acetate in 250 c. c. of water is'addedwhile mixing thoroughly. A precipitate forms.

The mixture is heated, while'being stirred, to a temperature of from 50.' C. preferablyabout 90 C. until a sample no longer blackens with ammonium sulphide;Thetprecipie tate is filtered, washed, dried-and treated with somewhat less alkali solution than is suflicent to efiectlcompl'ete solution, the solution is filtered and evaporated to'dryness.

Instead of 32 ii grams of mercuric acetate,

64 grams may be used, thus giving a ratio "of two atoms ofjmercury, instead of one'atom, to each molecule of halogenated fiuorescein.

Ewampze 58.4 grams (01 gram molecule) of Ot dl in 2000. c. of-

until a sample no longer blackens with am monium sulphide. It'is then filtered, washed, dissolved in an amount of alkali solution somewhat less than suflicient to eifect complete solution, and the solution is evaporated to dryness, preferably under reduced pressure. In place of 32 g. of mercuric acetate,

64 grams maybe used in 50O c. c. of water,

' the process being otherwise the same.

E mpze [H 64.8 grams (0.1 gram molecule) of ,B-tetra bromefluor escein are dissolved" by the addition of 200'c. c. normal alkali, diluted to l litre, and 21.6 grams (0.1 gram molecule) of yellow mercuricoxide are added. With constant stirring the mixture is heated at a temperature of SO-95C. until the oxide is dissolved, the solution is filtered and evapo- 1 rated to'dryness, preferably under reduced pressure. Instead of 21.6 grams of mercuric oxide two, three or four "times-this amount may be used.-

Example I V i 90 grams (0.1 gram molecule) of a-di: iodo ,8 tetrabrom-fluorescein are dissolved with 250 c. 0. normal alkali, diluted to 1 litre, and reprecipitated with 25 c. c. of glacial acetic acid mixed with 50 c'.-c. of water, with vigorous stirring. A solution of 32 grams of mercuric'acetate (0.1 gram molecule) in 250 c. c. of water is mixed thoroughly with the suspension, and the whole is heated to boiling until a sample no longer blackens with ammonium sulphide. It is then filtered, washed, dissolved in an" amount of alkali solution, slightly less than suflicient to effect complete solution, and the filtered solution is evaporated to dryness, preferably under reduced pressure. p

Twice the above amount of mercuric acetate may be used, the procedure being otherwise the same.

In the appended claim, the expression mercury derivatives embraces and is intended to embrace all possible mercury derivatives of the respective halogenated fluoresceins, and the free acids thereof as well as the alkali metal salts of the free acids.

The mercury introduced into the halogenated fiuorescein molecule probably exists as a part of a group comprising also a negative atom or group such as OH, CH3COO or a halogen depending upon the compound of mercury employed in the mercuration. The

references to mercury throughout the specification and appended claim will be understood to embrace such mercury containing groups. v e

40 I claim:

The mercury containing compounds of a-dihalogeno 8-tetrahalogeno flu o r e sc ein s, having the general formula:

. z oz wherein Z represents halogen, and a represents hydrogen, halogen or mercury, two'ms being halogenand at least one and not more 6Q than two ws being mercury. i V

In testimony whereof, I affix my signature.

EDWVIN 0. WHITE, 

